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Viral Production & Use

Biosafety

Requestor is responsible for compliance with
their institution's biosafety regulations.
Lentivirus is generally considered BSL-2. AAV is
generally considered BSL-1, but may require
BSL-2 handling depending on the insert.
Biosafety Guide

Resource Information

Viral Quality Control

Titering Method:

Colony formation assay: A549 cells were transduced with serial dilutions of 63593-LV and treated with blasticidin. Blasticidin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet. and counted.

Notes:

PCR confirmation of insert: PCR was carried out on the viral preparation with primers targeting Cas9 and the blasticidin-resistance gene. The PCR product was visualized on an agarose gel for size confirmation.
Forward Primer: SpCas9-F2 GATGAAGAACTACTGGCGGC
Reverse Primer: Blast-R GCTCTTTCAATGAGGGTGGA

Confirmation of protein expression: A549 cells were transduced with serial dilutions of 63593-LV and treated with blasticidin. Polyclonal pools of blasticidin-resistant cells were expanded, collected, lysed and tested for Cas9 expression via immunoblotting. You can view the stable cell line expression data here or in the image section at the top of this page. Read our protocol for generating stable cell lines here.

Addgene Comments

While the typical yield for lentiviral vectors ranges from 10⁶-10⁷ TU/mL, titers for large or toxic inserts, such as for Cas9, can be 10-fold to 100-fold lower. Scientists generating their own lentiviral particles from Cas9 should expect similarly low titers.

These plasmids were created by your colleagues. Please acknowledge the
Principal Investigator, cite the article in which the plasmids were described,
and include Addgene in the Materials and Methods of your future publications.